Hematopoiesis is regulated by the direct interaction between the group of hematopoietic stem cells having self-renewing capacity, hematopoietic progenitors supplied from hematopoietic stem cells and destined to differentiate in a predetermined direction and cells at various stages of continuous differentiation from the former to the latter, and supporting stroma cells as a hematopoietic micro-environment that surrounds those sets of cells, or by the indirect interaction between the first mentioned group of cells and humoral hematopoietic regulating factors secreted from the stroma cells. A large number of cytokines have been shown to participate in the growth and/or differentiation of hematopoietic stem cells into various mature blood cells via hematopoietic progenitors.
The development of genetic engineering has witnessed the gene cloning of the cytokines mentioned above and their industrial production has also become possible by genetic recombination technology. Among genetic recombinant hematopoietic factors are the granulocyte colony-stimulating factor (hereunder abbreviated as G-CSF) and the macrophage colony-stimulating factor (hereunder abbreviated as M-CSF) which are clinically applied as therapeutics of hematopoietic hypofunction (e.g. neutropenia) due to radiation exposure or chemotherapy, as well as erythropoietin (hereunder abbreviated as EPO) which is clinically applied as a therapeutic of renal anemia. However, the treatment with these hematopoietic factors simply leads to a temporary recovery of mature blood cells.
Hence, auto- or homo-grafting of hematopoietic stem cells has been performed as a means of treatment for fundamental improvement of hematopoietic function. Recently, peripheral hematopoietic stem cells transplantation has been spread rapidly and umbilical cord blood hematopoietic stem cells transplantation is drawing attention. However, they also involve many problems and, in particular, the rarity of hematopoietic stem cells in blood cells impose substantial burden on the donor and/or the recipient. It is therefore necessary to establish a method for ex vivo expansion of hematopoietic stem cells. Patients with lethal hereditary diseases, certain malignant tumors and AIDS, which currently have no effective methods of treatment, are being subjected to trials of gene therapy for complementing deficient or mutated genes (Juya Ohashi, Jikken Igaku, 12:333, 1994).
Hematopoietic stem cells, being capable of long time survival, are considered optimal target cells in gene therapy of the kind just described above. However, in order to achieve efficient transfection or infection with a retrovirus vector incorporating a desired gene, it is usually required that a small number of hematopoietic stem cells in the resting phase be put into the cell cycle and proliferated. Studies have been made on the effect of a stem cell factor (SCF) and flk-2/flt-3 ligand which are considered to participate in the growth of hematopoietic stem cells and hematopoietic progenitors.
It has been revealed by experimental studies that the c-kit/SCF signal is important for the growth of hematopoietic stem cells and hematopoietic progenitors (Blood, 78:1-19, 1991; Blood, 81: 2844-2853, 1993; Blood, 90:4767-4778, 1997) and the stem cell factor (SCF) has been shown to be a ligand for the c-kit which is the tyrosine kinase type receptor expressed in hematopoietic stem cells and hematopoietic progenitors (Cell, 63:167-174, 1990; Cell, 63:195-201, 1990; Cell, 63:225-233, 1990), leading researchers to anticipate that SCF may have effect on the growth of hematopoietic stem cells and hematopoietic progenitors. However, the c-kit is expressed only weakly on human hematopoietic stem cells and hematopoietic progenitors (Blood, 87:4136-4142, 1996) and SCF if used alone has low expansion activity and is not fully effective for the growth of hematopoietic stem cells and hematopoietic progenitors.
flk-2/flt-3 is a receptor type tyrosine kinase with recognized gene expression in various tissues and, in blood cells, is dominantly expressed in undifferentiated hematopoietic stem cells, with the flk-2/flt-3 ligand (FL) having been identified as a factor stimulating the growth of undifferentiated hematopoietic cells (Lyman, S. D. Curr. Opin. Hematol., 1998; 5(3): 192-6). But then this molecule, if used alone, has low expansion activity and is not fully effective for the growth of hematopoietic stem cells and hematopoietic progenitors.
Thus, neither SCF nor FL is fully effective for the growth of hematopoietic stem cells and hematopoietic progenitors if they are used singly, so combining them with various cytokines is considered ideal as a method of expansion hematopoietic stem cells and hematopoietic progenitors (Blood, 89: 2644-2653, 1997; Cancer Chemother. Pharmacol., 38[Suppl.]:64-68, 1996) and a study has been made on combining these molecules with TPO (thrombopoietin), interleukin 6 (IL-6)/soluble interleukin 6 receptor complex, Hyper IL-6 (fusion protein from IL-6 and IL-6 receptor), etc. (Exp. Hematol. 29:822-832, 2001). Further, it is desired to elucidate and obtain a new factor having stronger expansion activity.
Cofilin is a protein having molecular weight of about 19,000 and a member of actin-binding proteins (ABP) that bind to actin filaments (F-actin) at a molar ratio of 1:1 in response to a variety of signals, thus regulating the physical conditions of actin and performing primary function in the reconstitution of the actin-based cytoskeleton (Jikken Igaku, Vol. 12, No. 4:24-28, 1994). It is known that Cofilin, by binding to G-actin and cutting G-actin (actin monomer) and depolymerizing it, controls many cell responses including changes in shape, movements (motion), division, secretion, phagocytic (pinocytic) action, various signal transductions, etc. (Seikagaku, Vol. 71, No. 2:101-114, 1999). Cofilin in a cell occurs in both a phosphorylated and a dephosphorylated form and their activity for binding to actin is suppressed by phosphorylation but promoted by dephosphorylation.
It has recently become known that Cofilin is dephosphorylated in response to various external stimulations including the stimulation of platelets by thrombin, the stimulation of thyroid cells by a thyroid-stimulating hormone, the stimulation of parotid cells by isoproterenol and the stimulation of astrocytes by dibutyryl cAMP (Moon, A. & Drubin, D. G. (1995) Mol. Biol. Cell. 6, 1423-1431). It has also been reported that Cofilin is dephosphorylated as neutrophils or T cells are activated.
Methods have been proposed that are intended to treat certain kinds of disease either through the function of actin binding proteins (ABPs) or by regulating the ABP function. Included among such methods are those for treatment or disease alleviation by administering ABP to morbid tissues or organs resulting from actin deposits (JP 5-50603 A and JP 8-510998 A), as well as therapeutics for a variety of failure-to-control-apotosis associated diseases that act under the mechanism of apotosis modulation by suppressors of dephosphorylation of Cofilin (JP 10-67662 A and JP 10-87484 A).
However, to date there has been no report of Cofilin and their analogous compounds participating in the growth and/or differentiation of hematopoietic stem cells and/or hematopoietic progenitors.
An object of the present invention is to provide promoters of the growth and/or differentiation of hematopoietic stem cells and/or hematopoietic progenitors that are useful as therapeutics of diseases that develop from insufficient growth and/or differentiation of hematopoietic stem cells and/or hematopoietic progenitors, in particular, panhematopenia and/or diseases that are accompanied by hematopoietic hypofunction. Another object of the present invention is to provide a method of expanding hematopoietic stem cells ex vivo which comprises administering the promoters of the growth and/or differentiation of hematopoietic stem cells and/or hematopoietic progenitors, which method is also useful in transplantation of hematopoietic stem cells, gene therapy and regenerative medicine.